1 / 24

1- Making Density Gradients

1- Making Density Gradients. Pre-formed discontinuous gradients Pre-formed continuous gradients Self-generated gradients. 2- Density Gradient Harvesting. C. A. B. Discontinuous gradient by overlayering. A. B. C. D. Discontinuous gradient by underlayering. density. top. bottom.

montana
Download Presentation

1- Making Density Gradients

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 1- Making Density Gradients • Pre-formed discontinuous gradients • Pre-formed continuous gradients • Self-generated gradients 2- Density Gradient Harvesting

  2. C A B Discontinuous gradient by overlayering

  3. A B C D Discontinuous gradient by underlayering

  4. density top bottom Diffusion of a discontinuous gradient

  5. Discontinuous Continuous 45-60 min Rapid formation of continuous from discontinuous gradient

  6. A B T SB SB P M Two-chamber device for continuous gradients

  7. B The Gradient Master

  8. Density (g/ml) 2 min 1.13 2.5 min 3.0 min 1.11 1.09 1.07 1 3 5 7 9 Fraction Number Gradient Master profiles from 10% and 40% iodixanol at 80° and 20 rpm: effect of time

  9. axis of rotation g At rest Spinning g Swinging-bucket rotor

  10. Fixed-angle Vertical g 1g Fixed-angle and vertical rotors

  11. Swinging-bucket Axis of rotation Vertical Sedimentation path length of rotors

  12. A B Axis of rotation Sample/gradient in vertical rotor

  13. time g Self-generated gradients • At 360,000gav iodixanol molecules will start to sediment

  14. 350,000g 350,000g 1g 1g Self-generated gradient strategy

  15. Near-vertical rotors are best of all

  16. Iodixanol self-generated gradient requirements • Rotors with a short sedimentation path length ~ 17 mm • RCF of 180,000-350,000gav

  17. Density (g/ml) 15% iodixanol 1.30 1 h 3 h 1.25 20% iodixanol 1.20 1 h 3 h 1.15 1.10 1.05 1.00 1 3 5 7 9 11 13 15 Fraction Number Effect of time on iodixanol self-generated gradient in the Beckman TLN100 at 365,000g

  18. 1.3 15% (170,000g) 30% (170,000g) 15% (353,000g) 1.2 30% (353,000g) Density (g/ml) 1.1 1.0 1 3 5 7 9 11 Fraction Number Effect of RCF and [iodixanol] in Beckman VTi65.1 (3 h)

  19. Removal of banded material using a syringe

  20. Collecting a band from a sealed tube

  21. Gilson Fraction Collector P 96-well plate Harvest gradient into equal volume fractions by tube puncture

  22. Perfluorodecalin Perfluorodecalin P P Upward displacement

  23. P P P P M B EP C 1 2 3 4 Labconco Auto Densi-flow collects from meniscus

  24. MB Labconco Auto Densi-flow linked to a Gilson FC205 Fraction Collector

More Related